Flaviviruses and alphaviruses are serious human pathogens for which few vaccines or antiviral remedies are available. This Project confinues a successful collaboration with Project 3 on the structure and function of replicafion proteins with the goal of making fundamental discoveries about the mechanisms of polyprotein processing, replication ofthe plus-sense RNA genomes, and genome capping. Replicafion proteins carry out several catalyfic funcfions and thus are prime targets for development of antivirals. There is a dearth of the structural and biochemical data needed to bridge from the biological data to detailed molecular mechanisms, due primarily to the great difficulty of obtaining purified, active viral replicafion proteins. Our approach is to develop materials suitable for in vitro study, to determine crystal structures of proteins and of protein-protein or protein-RNA complexes, to assay biochemical funcfion, and in collaborafion with Project 3, to test hypotheses about molecular mechanism in in vivo assays. Areas of focus build on discoveries and materials made in the previous funding period. Specifically, these include the flavivirus protease-helicase in complex with its co-factor (NS2B-NS3) and RNA, the flavivirus RNA-dependent RNA polymerase and capping enzyme (RdRp, NSS), the alphavirus capping enzyme (nsPI), and the alphavirus RdRp (nsP4). Lower priority target molecules include the alphavirus helicase-protease (nsP2) and a protein of unknown function from each virus (flavivirus NS1 and alphavirus nsP3). The Project includes replicafion proteins from eight fiaviviruses (dengue types 2, 3 and 4, West Nile, yellow fever, Japanese encephalifis, Kyasanur Forest and fick-borne encephalifis viruses) and five alphaviruses (Sindbis, Semliki Forest, Venezuelan equine encephalitis, Aura and Ross River viruses). Nine of these viruses are on the NIAID Priority Pathogens list. Complefion ofthe proposed research will provide a strong structural basis for understanding key processes in the virus life cycle, as well as mechanistic hypotheses about individual replicafion steps. The project is highly synergisfic with Project 3 because biochemical mechanisms are rapidly tested in vivo, and biological observafions are rapidly dissected in terms of biochemistry and structure. RELEVANCE (See instructions): Flaviviruses and alphaviruses cause a variety of human diseases for which no treatments are available. This study of how the viruses reproduce will provide an understanding of crifical steps of the virus life cycle, which can be exploited to develop antiviral treatments.